Anemia is a common and serious side effect of cancer-chemotherapy, which is often treated with recombinant human erythropoietin (EPO) to increase red blood cell production. EPO stimulates erythropoiesis through binding to and activating its natural target, the human EPO receptor. In hematopoietic stem and progenitor cells, when EPOR is activated, the resulting signal transduction leads to erythroid differentiation and production of red blood cells. However, the EPOR is not only expressed in hematopoietic cells, but has also been detected in non-hematopoietic tissues and tumors that can therefore be stimulated by EPO. Since EPO has a pro-survival, anti-apoptotic effect, EPO can actually promote tumor progression if EPOR is expressed on the tumor cells or surrounding tissues. Because EPO is a soluble protein, it can travel throughout the body, where it can activate the EPOR wherever it is expressed. Currently, there are no treatment options other than EPO treatment and blood transfusions to increase red blood cell production. Therefore, a therapeutic that is able to activate the EPOR and stimulate erythropoiesis in a cell-autonomous manner would be extremely useful. Using genetic methods, we have isolated an artificial transmembrane protein that can activate the human EPOR and induce erythroid differentiation in the absence of EPO, and because it is expressed from within the target cells, it functions cell-autonomously. To aid in the design of better therapeutics based on this protein, this study aims to determine the mechanism of activation of this small transmembrane protein to gain a better understanding of how it functions and interacts with the human EPOR. Through comprehensive mutational and biochemical analyses, I will determine the critical residues of both the EPOR and transmembrane protein that are required for their interaction and activity. Additionally, this work has the potential to inform our understanding of the normal mechanism of EPOR activation and receptor conformation necessary to initiate signaling and drive red blood cell production.